Antibodies against phoshorylated VASP (vasodilator-stimulated phosphoprotein), hybridoma cells for their preparation, and their use

ABSTRACT

The invention relates to antibodies against VASP (vasodilator-stimulated phosphoprotein) which only bind VASP as an antigen when VASP is present in phosphorylated form, to hybridoma cells for their preparation, and to the use of the antibodies or antibody fragments as diagnostic agents and/or therapeutic agents.

This application claims the benefit of provisional application No.60/079,375, filed Mar. 26, 1998, the content of which is incorporatedherein by reference.

The invention relates to antibodies against VASP (vasodilator-stimulatedphosphoprotein) which only bind VASP as antigen when VASP is present inphosphorylated form, to hybridoma cells for their preparation, and tothe use of the antibodies or antibody fragments as diagnostic agentsand/or therapeutic agents.

Diseases of the vascular system are responsible for a large number ofchronic and life-threatening diseases, such as cardiac infarction,stroke, arterial occlusion disease and many forms of kidney failure.

The endothelial cells, which, inter alia, control the blood coagulationsystem, the functional state of the thrombocytes, the migration ofinflammatory and tumor cells into the vascular wall, the state ofcontraction and growth of smooth muscle cells and consequently alsoblood pressure and vascular wall structure and also neovascularization,are of particular importance for regulating the vascular system. Many ofthese vascular wall functions are disturbed in serious vasculardiseases, a situation which can in the end lead to cardiac infarction,stroke and many forms of kidney failure.

The endothelium forms the important substances prostacyclin (PGI₂) andnitrogen monoxide (NO), which is also known as endothelium-derivedrelaxing factor (EDRF), which substances inhibit both the thrombocytesand the vascular muscle cells. The endothelial functions are controlledby said endothelial factors such as prostacyclin (PGI₂) or EDRF, e.g.NO.

Consequently, in order to treat diseases which are associated with anendothelial dysfunction in a specific manner, it is necessary to developbiochemical parameters which enable an endothelial dysfunction to bediagnosed and its course to be controlled.

It is desirable to be able to recognize endothelial dysfunctions asearly as possible, that is at a stage at which irreversible damage, suchas atherosclerotic lesions, caused by endothelial dysfunctions has stillnot manifested itself.

Identification of an endothelial dysfunction at an early stage makes itpossible to develop new therapeutic approaches which can bring aboutreversible treatment of the endothelial dysfunction.

Methods which are known for determining in-vivo endothelial functionsare invasive detection methods, such as quantitative angiography, orelse non-invasive, image-providing methods. Disadvantages of thesemethods are that these investigations are carried out directly on thepatient, and are difficult to quantify and very expensive.

It is therefore also desirable to have availablebiochemical/immuno-biological methods which make it possible todetermine endothelial functions rapidly and simply in biologicalmaterial ex vivo, for example in cell samples or blood samples by meansof routine investigations in the laboratory. Endothelial functions arethose functions which can be regulated by endothelial factors such asprostacyclin (PGI₂) or EDRF, e.g. NO.

It is an object of the invention to provide reagents for the evaluationand modulation of endothelial function. According to this and otherobjects of the invention, an antibody is provided which is directed toVASP (Vasodilator-stimulated phosphoprotein) and binds VASP only whenVASP is phosphorylated.

In one aspect of the invention an antibody is disclosed which binds VASPonly when VASP is phosphorylated at position serine 239 (phosphoserine239 VASP). In another aspect an antibody is provided which binds VASPonly when VASP is phosphorylated at position serine 157 (phosphoserine157 VASP).

Different embodiments of the invention include polyclonal antibodies,monoclonal antibodies and antibody fragments. Other embodiments includea monoclonal antibody produced by the hybridoma cell line 16C2, andparticularly Mab 16C2.

It is a further object of the invention to provide hybridoma cells whichare useful in manufacturing the antibodies of the invention. Further tothis object of the invention hybridoma cell line is provided whichproduces a monoclonal antibody against VASP which binds VASP only whenit is phosphorylated. In one embodiment, the hybridoma cell line 16C2(DSM ACC2330) is provided. p It is yet another object of the inventionto provide methods of evaluating endothelial function. According to thisobject of the invention methods are provided for determining thephosphorylation status of VASP. In one aspect of the inventionbiological material is contacted with an antibody against VASP(vasodilator-stimulated phosphoprotein), which binds VASP as antigenonly when VASP is phosphorylated.

In another aspect of the invention a quantitative method is providedwhich further involves quantifying the amount of VASP antibody whichbinds the biological material. A specific embodiment includes a Westernblotting method, which entails resolving VASP by eletrophoresis andcontacting, the VASP with an antibody against VASP, which binds VASPonly when it is phosphorylated. Another embodiment is a flow cytometrymethod, which involves contacting a sample with an antibody against VASPwhich binds VASP only when it is phosphorylated.

In yet another aspect of the invention, methods of diagnosis areprovided. A representative method entails contacting a sample with anantibody against VASP which binds VASP only when it is phosphorylatedand evaluating the phosphorylation state of VASP. In one embodiment,this method involves quantitatively determining the phosphorylation ofVASP in the sample. In another embodiment, methods are described wherethe antibody binds phosphoserine 239 VASP or phosphoserine 157 VASP. Instill another embodiment, the sample is human thrombocytes or humanwhole blood.

It is yet another object of the invention to provide methods fordetecting markers of endothelial function. In one aspect, a method fordetecting substances which affect the level of cGMP and/or cAMP isprovided which involves testing a sample from patient who has beenexposed to a substance of affecting cGMP and/or cAMP levels, contactingthe sample with an antibody against VASP which binds VASP only when itis phosphorylated and evaluating the phosphorylation state of VASPrelative to a control sample.

In yet one more aspect, a method is detailed for detecting endothelialdysfunction which involves contacting a sample from a patient with anantibody against VASP which binds VASP only when it is phosphorylatedand evaluating the phosphorylation state of VASP relative to a normalcontrol sample.

It is a further object of the invention to provide a convenient kit foraccomplishing the diagnostic methods disclosed herein. Further to thisobject a diagnostic kit is provided which contains an antibody againstVASP which binds VASP only when it is phosphorylated.

It is still another object of the invention to provide methods oftreating patients suffering from endothetial dysfunction. According tothis and other a therapeutic objects of the invention a method oftreatment is disclosed where a patient in need of treatment isadministered a therapeutically effective amount of an antibody againstVASP which binds VASP only when it is phophorylated.

Human VASP (vasodilator-stimulated phosphoprotein), which isphosphorylated in thrombocytes andvascular wall cells in response tohypotensive (vasodilatory) hormones and drugs, has recently beendiscovered, isolated and characterized from the point of view ofmolecular genetics (Haffner et al., EMBO J. 14, 19-27, 1995).

VASP is an important component of the ANF/NO/cGMP/cGMP protein kinasesignal pathway, which is very important physiologically,pathophysiologically and pharmacologically, and also of the cAMP/cAMPprotein kinase signal pathway (U. Walter, Blick 1/97 WürzburgUniversity, pp. 79-81,1997). VASP is expressed in almost all human andanimal cells, with particularly high concentrations being found inthrombocytes, vascular smooth muscle cells and fibroblasts. In culturedcells, VASP is associated with focal contacts (cell/matrix contactsites), cell/cell contacts, microfilaments and dynamic membrane regions(e.g. leading edge) (Walter et al., Agents and Actions 45S, 255-268,1995).

The phosphorylation of VASP in the vascular system correlates with theinhibition of thrombocyte adhesion/aggregation, the inhibition of smoothmuscle contraction/migration and the inhibition of paracellularendothelial permeability.

VASP is phosphorylated and dephosphorylated at three different sites(serine 157, serine 239 and threonine 278, see Horstrup et al., Eur. J.Biochem. 225, 21-27 (1994)). Serine 239 is occasionally also designatedSerine 238, specifically when the first methionine of VASP is notincluded in the count.

VASP serine 239 is phosphorylated in intact human vascular cells(thrombocytes, endothelial cells and smooth muscle cells) in response tophysiological and pharmacological NO donors and thrombocyte inhibitorsand vasodilators.

The phosphorylation of VASP serine 239 is mediated, in particular, bycGMP-dependent protein kinases which are activated, by way of the cGMP,by important hormones such as natriuretic peptides or NO-releasingsubstances and drugs. Phosphorylation of VASP serine 239 is additionallymediated by cAMP-dependent protein kinases which are activated by way ofcAMP-increasing hormones and drugs.

While cAMP-dependent protein kinases principally phosphorylate theserine 157 position, they also phosphorylate the serine 239 position ofthe VASP. VASP phosphorylation at the serine 157 position has also beenobserved to be closely correlated with inhibition of the binding offibrinogen to glycoprotein IIb-IIIa in human blood platelets (Horstrupet al., Eur. J. Biochem. 225, 21-27 (1994))

Determination of the degree to which VASP was phosphorylated inbiological material, for example in extracts of tissues and cells, inparticular determination of the phosphorylation of VASP at position 239and/or position 157, would be an important biochemical parameter whosemeasurement would make it possible to develop a diagnostic system fordetecting all cGMP-increasing and/or cAMP-increasing hormones or drugs,such as atrial natriuretic factor (ANF), guanylin, NO-releasingsubstances and drugs, and furthermore enable conclusions to be drawnwith regard to in-vivo endothelial functions.

Antibodies that specifically bind to reversibly phosphorylated proteinshave been described in U.S. Pat. No. 5,599,681, WO93/21230 and U.Walter, Blick 1/97 Wüjrzburg University, pp. 79-81,1997.

The object of the present invention was to developbiochemical/immuno-biological methods which enable the phosphorylationstatus of VASP in biological material to be determined qualitativelyand/or quantitatively in a rapid in simple manner.

This object was achieved by the provision of antibodies which only bindVASP as antigen when the VASP is present in phosphorylated form.

The invention consequently relates, in a quite general manner, toantibodies which only bind VASP as antigen when VASP is present inphosphorylated form.

Within the meaning of the invention, antibodies are to be understood asbeing both polyclonal antibodies and monoclonal antibodies (Mabs) and atheir fragments, and also SCF fragments or other synthetic orrecombinant protein domains, which specifically recognize phosphorylatedregions in the VASP.

Fragments of antibodies include any portion of the antibody which iscapable of binding the target antigen, in this case VASP or a specificportion thereof. Antibody fragments specifically include F(ab′)₂, Fab,Fab′ and Fv fragments. These can be generated from any class ofantibody, but typically are made from IgG or IgM. They may be made byconventional recombinant DNA techniques or, using the classical method,by proteolytic digestion with papain or pepsin. See CURRENT PROTOCOLS INIMMUNOLOGY, chapter 2, Coligan et al., eds., (John Wiley & Sons1991-92).

F(ab′)₂ fragments are typically about 110 kDa (IgG) or about 15G kDa(IgM) and contain two antigen-binding regions, joined at the hinge bydisulfide bond(s). Virtually all, if not all, of the Fc is absent inthese fragments. Fab′ fragments are typically about 55 kDa (IgG) orabout 75 kDa (IdM) and can be formed, for example, by reducing thedisulfide bond(s) of an F(ab′)₂ fragment. The resulting free sulfhydrylgroup(s) may be used to conveniently conjugate Fab′ fragments to othermolecules, such as detection reagents (e.g., enzymes). Fab fragments aremonovalent and usually are about 50 kDa (from any source). Fab fragmentsinclude the light (L) and heavy (H) chain, variable (V_(L) and V_(H),respectively) and constant (C_(L)C_(H), respectively) regions of theantigen-binding portion of the antibody. The H and L portions are linkedby an intramolecular disulfide bridge.

Fv fragments are typically about 25 kDa (regardless of source) andcontain the variable regions of both the light and heavy chains (V_(L)and V_(H), respectively). Usually, the V_(L) and V_(H) chains are heldtogether only by non-covalent interacts and, thus, they readilydissociate. They do, however, have the advantage of small size and theyretain the same binding properties of the larger Fab fragments.Accordingly, methods have been developed to crosslink the V_(L) andV_(H) chains, using, for example, glutaraldehyde (or other chemicalcrosslinkers), intermolecular disulfide bonds (by incorporation ofcysteines) and peptide linkers. The resulting Fv is now a single chain(i.e., SCFv).

One preferred method involves the generation of SCFvs by recombinantmethods, which allows the generation of Fvs with new specificities bymixing and matching variable chains from different antibody sources. Ina typical method, a recombinant vector would be provided which comprisesthe appropriate regulatory elements driving expression of a cassetteregion. The cassette region would contain a DNA encloding a peptidelinker, with convenient sites at both the 5′ and 3′ ends of the linkerfor generating fusion proteins. The DNA encoding a variable region(s) ofinterest may be cloned in the vector from fusion proteins with thelinker, thus generating an SCFv.

In an exemplary alternative approach, DNAs encoding two Fvs may beligated to the DNA encoding the linker, and the resulting tripartitefusion may be ligated directly into a conventional expression vector.The SCFv DNAs generated any of these methods may be expressed inprokaryotic or eukaryotic cells, depending on the vector chosen.

Preference is given to monoclonal antibodies and their fragments whichonly bind VASP as antigen when VASP is present in phosphorylated form.

Particular preference is furthermore given to monoclonal antibodies andtheir fragments which only bind VASP, or peptides which encompass thepeptide sequence around serine 239 of VASP, as antigen when the serineat position 239 is phosphorylated (phosphoserine 239 VASP).

Within the meaning of the invention, the term VASP is also to beunderstood as meaning derivatives of VASP, i.e. functionally equivalentmoieties, mutants, fragments or variants of VASP, for examplephospho-serine 239 VASP which is additionally phosphorylated at positionserine 157 and/or threonine 278, and also, for example, glycosylationmutants and other covalent modifications and structural elements whichare of importance for protein/protein interaction. Within the meaning ofthe invention, the term VASP encompasses both human VASP and VASP fromother species, in particular from mammals such as rat, mouse, rabbit,dog, pig or monkey. Antibodies may be produced by conventional methodsfor instance according to the technique described by Köhler and Milstein(Köhler and Milstein, Nature 256; 495, 1975). Typically, whereselectivity based on phosphorylation state is desired, antibodies areraised against the phosphorylated forms of VASP. The antibodies isolatedmay be screened using conventional methods to ascertain selectivity.Preferably, antibodies are raised against peptide fragments of VASPwhich contain serine 157, serine 239 and/or thereonine 278.

The length of the antigenic VASP peptide is unimportant, so long as itis capable of eliciting a humoral response. Typical antigenic peptidesare less than about 50 amino acids long. Some preferred peptides areless than about 20 amino acids in length. Most preferred peptides arebetween about 6 and about 12 amino acids long. Preferred peptidesinclude KLRKVS²³⁹ KQ or RKVS²³⁹ KQE. The peptide preferably isphosphorylated at serine 157, serine 239 and/or threonine 278. Peptidesmay be obtained by recombinant means. Typically, they are producedeither by proteolytic degradation of VASP or in vitro synthesis andphosphorylation.

Preference is furthermore given to monoclonal antibodies which exhibitthe abovementioned properties and which can be employed in flowcytometry, This requires the specificity of the novel antibodies to beretained even when the antigen is subjected to conditions which mayalter the conformation of the antigen, as is to be expected in thefixation steps which are customarily employed in flow cytometry. Thenovel antibodies can, for example, be examined for their suitability foruse in flow cytometry simply by means of testing them out.

The monoclonal antibody 16C2 is particularly preferred.

The novel antibodies may be prepared using methods which are known perse to the skilled person. The preparation of hybridoma cells using thetechnique described by Köhler and Milstein (Köhler and Milstein, Nature256; 495, 1975) may, in particular, be mentioned for preparingmonoclonal antibodies. The specificity of the purified antibodies can bechecked, for example, using the following test methods:

a) Western blotting using recombinant VASP which is phosphorylated bycAMP-dependent or cGMP-dependent protein kinases to differing extents,in association with which it must only be possible to observe a positivesignal with phosphorylated VASP.

b) Western blotting using extracts of cells, for example Pkt2 cells,which have been transfected with human VASP or with VASP which has beenmutated in different ways and in which in each case one of the threephosphorylation sites has been mutated and thereby eliminated (S157A)VASP, (S239A) VASP and (T278A) VASP), and, in each case, human cGMPprotein kinase. Those antibodies are suitable in which the positivesignals in the Western blot are eliminated by the S239A mutation but notby the other two mutations.

c) Western blotting using human thrombocytes which have been treatedwith different vasodilators (e.g. prostacyclin or NO donors) oractivators of cAMP protein kinase or cGMP protein kinase. The antibodyis then suitable when a specific band is detected in the extract onlywith phosphorylated VASP. The antibodies can also be screened in ananalogous manner using human fibroblasts and also rat and mousethrombocytes.

d) Immunofluorescence investigations using fixed human thrombocytes andendothelial cells. A positive signal is observed when VASP is present asa phosphoprotein.

The invention furthermore relates to hybridoma cell lines which producenovel monoclonal antibodies, with the hybridoma cell line 16C2, whichproduces the monoclonal antibody 16C2, being particularly preferred.

The hybridoma cell line 16C2, which produces the monoclonal antibody16C2, has been deposited in the Deutsche Sammiung von Mikroorganismenund Zelikulturen (German collection of microorganisms and cellcultures), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, inaccordance with the rules of the Budapest treaty on Nov. 6, 1997 underthe following number: DSM ACC2330.

The novel antibodies are suitable for qualitatively and/orquantitatively, preferably quantitatively, determining both thephosphorylation of VASP which occurs under in-vitro conditions and thatwhich occurs under in-vivo conditions, preferably for determining theserine 239 and/or serine 157 phosphorylation of VASP, particularlypreferably for determining the serine 239 phosphorylation of VASP.

Quantitative methods of using antibodies are well known in the art andmay be found, for example, in CURRENT PROTOCOLS IN IMMUNOLOGY, supra.These methods include enzyme-linked immunosorbant assays (ELISA)radioimmunoassays (RIAs) and the like.

The novel antibodies are furthermore suitable for qualitatively and/orquantitatively, preferably quantitatively, determining phosphorylatedVASP, preferably phosphoserine 239 VASP and/or phosphoserine 157 VASP,particularly preferably phosphoserine 239 VASP, in biological material.

Biological material is, for example, understood as being: cell extracts,tissue extracts, cell slices, cell tissue and cells, such asthrombocytes, leukocytes, endothelial cells, smooth muscle cells andfibroblasts. The biological material can be derived from humans or elsefrom other mammals such as rat, mouse, rabbit, dog, pig or monkey.Biological material of human origin is preferred.

The novel antibodies can be used to quantitatively determinephosphorylated VASP, in particular phosphoserine 157 VASP and/orphosphoserine 239 VASP, in biological material by means ofquantitatively evaluating autoradiograms of Western blots in accordancewith methods which are known to the skilled person, for example usingNIH gel blotting Image 1.6 software, or else by means ofimmunofluorescence.

Due to the correlation which exists between the phosphorylation ofserine 239 and serine 157 of VASP and the activity of cGMP-dependentand/or cAMP-dependent protein kinases, the novel antibodies are alsosuitable for use as agents for diagnosing cGMP signal pathways and cAMPsignal pathways, in particular for diagnosing cGMP signal pathways.

The use of the novel antibodies to determine phosphoserine 239 VASP inbiological material by means of the Western blotting technique andimmunofluorescence also makes it possible to develop diagnostic methodsfor detecting cGMP-increasing substances, hormones or drugs. Exampleswhich may be mentioned are: ANF, guanylin, NO-releasing substances ordrugs. For example, control of the course and the therapy of NO donorscan be monitored, which, inter alia, provides information on any nitratetolerance phenomena which may develop.

Due to the correlation which exists between the phosphorylation ofserine 157 of VASP and the activity of cAMP-dependent protein kinases,the novel antibodies can also be employed as agents for diagnosingcAMP-increasing substances, hormones or drugs.

The invention consequently also relates to the use of the novelantibodies or fragments thereof in diagnosis and/or therapy.

The novel diagnostic methods are methods which can be carried out in thelaboratory, outside the human body (ex vivo).

The novel antibodies can be used for determining the degree ofphosphorylation of VASP in biological material which derives fromdifferent species. Examples which may be mentioned are: man, rat, mouse,rabbit, dog, pig or monkey. Preference is given to using the novelantibodies for determining phosphorylated VASP in biological materialfrom man, rat, mouse or dog. Particular preference is given to using thenovel antibodies for determining phosphorylated VASP in human biologicalmaterial.

The novel antibodies or fragments thereof can also be used inbiosensors. Biosensors are known per se to the skilled person.Particular preference is given to a method which uses a second specificbinding partner, such as an antibody, a lectin or a receptor. Fordetection and quantification in this case, one of the specific bindingpartners can carry a detectable label. These labels are known per se tothe skilled person and can, for example, be a chromophore, aluminophore, a fluorophore, an enzyme, a radioactive isotope or acolored or colorless particle. Preference is given to a method in whichthe unlabeled, specific binding partner is coupled directly orindirectly, for example by way of another antibody or a biotinlavidinbridge, to a solid phase using methods known per se to the skilledperson.

The novel antibodies or their fragments can also be radioactivelylabeled by methods known to the skilled person so that they can be usedfor immunoscintigraphy or else for immunotherapy. In addition, thesemonoclonal antibodies can be used as active compound carriers andemployed for the therapy of diseases which are caused by endothelialdysfunctions.

Following analysis of the complete nucleotide sequence of the V genes ofMab 16C2, it is also technically possible to produce antibody constructsby, for example, inserting the hypervariable regions into a human Mabskeleton (Jones et al., Nature 321, 522-525, 1986; Verhoyen et al.,Science 239,1534-1536,1988).

Preference is given to quantifying the antigen-bound antibody in bloodplatelets by means of flow cytometry, for example carried out on wholeblood samples which have, where appropriate, been fixed using methodsknown to the skilled person. Methods for flow cytometry are known to theskilled person and can be carried out as described, for example, in G.Otten and W. M. Yokoyama (1992) Flow cytometry analysis using the BectonDickinson FACScan, Current Protocols in Immunology, 5.4.1-5.4.19.

The flow-cytometric analysis of VASP phosphorylation, in particular VASPserine 239 phosphorylation, using the novel antibodies is not restrictedto human thrombocytes and can also be carried out on other cell typessuch as lymphocytes, monocytes, leukocytes, endothelial cells and smoothmuscle cells.

The use of flow cytometry to determine the phosphorylation of VASP, inparticular of phosphoserine 239 VASP, in freshly withdrawn humanthrombocytes with the aid of the novel antibodies makes it possible todetermine ex vivo the in vivo activity of endothelial factors such as NOand prostacyclin and consequently to assess endothelial function orendothelial dysfunction in cardiovascular disorders as are found, forexample, in arteriosclerosis, hypertension, diabetes, cardiacinsufficiency and renal insufficiency.

Determining VASP phosphorylation, in particular VASP serine 239phosphorylation, with the aid of the novel antibodies also makes itpossible to control the therapy of the abovementioned diseases. Thedevelopment of resistances to therapy, such as nitrate tolerance, canalso be determined.

The novel antibodies can also be employed as therapeutic agents in thatthey affect the phosphorylation status of VASP, and/or its interactionswith proteins, in biological material.

The invention consequently also relates to pharmaceutical preparationswhich comprise at least one of the novel antibodies.

The novel preparations may be used enterally (orally), parenterally(intravenously), rectally or locally (topically). They can beadministered in the form of solutions, powders (tablets or capsulesincluding microcapsules), liposome preparations, lipid complexes,colloidal dispersions, injection solutions or suppositories. Thepharmaceutically customary liquid or solid fillers and extenders,solvents, emulsifiers, glidants, taste corrigents, dyes and/or bufferingsubstances are suitable auxiliary substances for formulations of thisnature. 0.1-100 mg are administered per kg of body weight as anexpedient dose. They are expediently administered in dosage units whichat least comprise the effective daily quantity of the novel antibodies,e.g. 30-3000 mg.

The daily dose which is to be administered depends on the body weight,age, sex and condition of the mammalian subject. However, higher orlower daily doses may also be called for. The daily dose can either beadministered by being given on one occasion in the form of a singledosage unit or in the form of several smaller dosage units, or elsebeing given on several occasions, at predetermined intervals, in theform of subdivided doses.

In order to produce pharmaceutical preparations, the novel antibodiescan be worked into therapeutically inert organic and inorganicexcipients. Lactose, corn starch or derivatives thereof, tallow andstearic acid or salts thereof are examples of such excipients fortablets, coated tablets and hard gelatin capsules. Water, polyols,sucrose, invert sugar and glucose are suitable excipients for preparingsolutions. Water, alcohols, polyols, glycerol and vegetable oils aresuitable excipients for injection solutions. Vegetable oils and hardenedoils, waxes, fats and semisolid polyols are suitable excipients forsuppositories. The pharmaceutical preparations can also comprisepreservatives, solvents, stabilizers, wetting agents, emulsifiers,sweeteners, dyes, flavorings, salts for altering the osmotic pressure,buffers, coating agents, antioxidants and, where appropriate, othertherapeutic active compounds.

The invention also relates to a process for preparing a novelpharmaceutical, wherein at least one of the novel antibodies is broughtinto a suitable administration form together with a pharmaceuticallysuitable and physiologically tolerated excipient and, where appropriate,other suitable active compounds, additives or auxiliary substances.

The invention furthermore relates, quite generally, to a diagnosticprocess for determining the phosphorylation of VASP, preferably theserine 239 phosphorylation of VASP, with the aid of other specificprobes such as polygonal antibodies, SCFV fragments or other syntheticor recombinant protein domains which specifically recognizephosphorylated sequences in VASP, in particular in phosphoserine 239VASP.

The invention furthermore also relates to a diagnostic process fordetermining the serine 157 phosphorylation and/or threonine 278phosphorylation of VASP With the aid of specific probes such asmonoclonal antibodies, polyclonal antibodies, SCFV fragments or othersynthetic or recombinant protein domains which specifically recognizesequences in VASP which contain phosphorylated serine 157 and/orthreonine 278.

By means of binding to proline-rich peptides, such as zyxin andvinculin, and at the same time binding its own proline-rich amino aciddomain to profilin, VASP makes possible the formation of filamentousactin, with the VASP functioning as an adapter molecule. Disturbance ofthis protein/protein interaction can lead to faulty thrombocyteaggregation or vascular contraction. Thus, the formation of actin/myosinbridges is a prerequisite for the contraction of smooth muscle cells,for example. It has been found that the phosphorylation of VASP at theserine 157 position correlates with inhibition of the fibrinogenreceptor glycoprotein IIb/IIIa (Horstrup et al., Eur. J. Biochem., 225,21-27, 1994). Determination of VASP phosphorylation at the serine 157position consequently makes it possible to search systematically forsubstances or drugs which affect the interaction of VASP with itsintracellular binding partners and are, for example, suitable for thetherapy of cardiovascular diseases which are associated with vasculardamage.

Particular preference is also given to the embodiments which aredescribed in the implementation examples.

The following examples serve to clarify the invention and do notrestrict it in any way.

EXAMPLES Example 1 Isolation of a Monoclonal Antibody AgainstPhosphoserine 239 VASP

A phosphorylated peptide and a non-phosphorylated peptide, each of whichencompasses the peptide sequence KLRKVS²³⁹ KQ or RKVS²³⁹ KQE aroundserine 239, are synthesized using an Applied Biosystems peptidesynthesizer (Model 431A) in accordance with the Fmoc chemistry which isfamiliar to the skilled person. The phosphoserine in the phosphorylatedpeptide is incorporated during peptide synthesis using Fmoc-serine[PO(Obzl)OH—OH] from Calbiochem. MS-confirmed peptides are purifiedusing RPC and a VYDAC 218TP column (purity>98%).

After having been activated with bromoacetic acid orbromoacetic-N-hydroxysuccinimide ester (Sigma), the peptides which havebeen prepared in this way are conjugated to thiolated KLH (keyholelimpet hemocyanin, nanoTools). Female Balb/c mice (6 weeks old) areimmunized subcutaneously 4×at 14-day intervals with theKLH-phosphopeptide (10 μg/mouse) containing complete Freund's adjuvantin the first injection and incomplete adjuvant in the 3 followinginjections. The mice are then (2 weeks later) given booster injectionsof 10 μg of immunogen in PBS (phosphate-buffered saline) on threeconsecutive days. 1 day after the last booster injection, the mice aresacrificed and the spleens removed. Spleen cells are isolated and fusedwith non-producing myeloma cells (e.g. PAI-Zellen, J. W. Stockeret al.(1982) Res. Disclosure, 21713) using the established Köhler/Milsteinmethodology.

A differential screening method using phosphorylated/non-phosphorylatedpeptide as well as phosphorylated/non-phosphorylated recombinant humanVASP is employed to test hybridoma cells for their ability to secreteantibodies against phosphoserine 239 VASP.

For the test using phosphorylated/non-phosphorylated peptide, thesepeptides are coupled covalently to DNA-BIND-ELISA plates (from Costar)and the hybridoma supernatents are screened using the ELISA method.

Supernatants which recognize phosphopeptide are additionally examinedfor their ability to recognize completely phosphorylated recombinant (E.coli system) human VASP but not the correspondingly dephosphorylatedVASP.

Monoclonal antibodies from the supematants of the hybridoma cells whichhave been identified by the above-described methods as being positivecan preferably be purified from serum-free hybridoma cell cultures bymeans of thiophilic adsorption chromatography (POROS 50-OH, nanoTools).

Using different test methods, it was possible to identify andcharacterize one of the isolated antibodies (clone 16C2) as being amonoclonal antibody of the mouse IgG1κ class which only recognizes VASPwhen this protein is phosphorylated at the serine 239 position. Antibody16C2 does not recognize other proteins and other VASP phosphorylationsites under the conditions employed.

Monoclonal antibodies which specifically recognize phosphoserine 157VASP or phosphothreonine 278 VASP can be isolated in analogy with theabove-described method by employing phosphorylated peptides whichencompass the known peptide sequences around serine 157 or threonine 278of the VASP protein, respectively, as the antigen.

The generation of monoclonal antibodies recognizing VASP phosphorylatedat Position Ser 239 is also described in Smolenski et al., J.

Biol. Chem., 237, 32, 20029-20035 (1998).

Example 2 Flow-cytometric Analysis of VASP Serine 239 Phosphorylation

Analysis of VASP Serine 239 Phosphorylation in Washed Human Thrombocytes

Human thrombocytes are prepared, and VASP phosphorylation is stimulatedby incubating with vasoactive substances, as described (Eigenthaler etal., Eur. J. Biochem., 205, 471-481, 1992). The reaction is stopped byfixing, at room temperature for 10 min, with formaldehyde at a finalconcentration of approx. 3.5%. After a washing procedure (optional), thecells are permeabilized with Triton X-100 and then washed. Staining iseffected by incubating with the appropriate antibody, which is eitherdirectly fluorescence-labeled or colored using a second antibody whichis fluorescence-labeled. Expediently, 0.5-5 μg, preferably 1-2 μg ofprimary antibody are employed per ml for this purpose. 1.7 μg/ml are,for example,. suitable when Mab 16C2 is used as the primary antibody.The binding of antibody 16C2 is then determined and analyzed in the flowcytometer as described, for example, in G. Otten and W. M.Yokoyama,(1992) Flow cytometry analysis using the Becton DickinsonFACScan, Current Protocols in Immunology, 5.4.1-5.4.19.

Example 3 Use of Western Blotting to Determine VASP Serine 239Phosphorylation in Cell Extracts

In parallel with the flow cytometry (Example 2), Western blotting isused to determine the phosphorylation of VASP in cell extracts by meansof methods which are familiar to the skilled person and which aredescribed, for example, in Eigenthaler et al. (Eur. J. Biochem., 205,471-481, 1992). The concentration of the antibody employed isexpediently between 0.1 and 5, preferably from 0.5 to 1.0, μg/ml,depending on the content of VASP in the sample to be determined. Forexample, for human thrombocytes and rat thrombocytes it is advantageousto use 0.5 μg of antibody/ml, while for human umbilical cord endothelialcells it is advantageous to use 1.0 μg of antibody/ml. In principle,analysis of VASP serine 239 phosphorylation in cell extracts by means ofWestern blot analysis and on fixed cells by means of flow cytometryyielded identical results.

Example 4 Analysis of VASP Serine 239 Phosphorylation in Whole Blood orPlatelet-rich Plasma (PRP)

Whole blood or PRP are incubated with vasoactive substances and theincubation is stopped by fixing, at room temperature for 10 min, withformaldehyde at a final concentration of approx. 3.5%. PRP is preparedfrom whole blood as described in Eigenthaler et al. (Eur. J. Biochem.,205, 471-481, 1992). Thrombocytes in the PRP are pelleted bycentrifugation and resuspended in physiological buffer (Eigenthaler etal., 1992, see above). The thrombocytes are permeabilized and thenstained as described above for washed thrombocytes.

Example 5

Time Course of the Stimulation of VASP Serine 239 Phosphorylation inWashed Human Thrombocytes Following Treatment with Sodium Nitroprusside(SNP)

Human thrombocytes were treated with 100 μM SNP. Cell samples werewithdrawn at times 0, 1, 3 and 5 minutes. The antibody 16C2 wars used todetermine the phosphorylation of VASP serine 239 in parallel by means ofthe Western blot analysis of extracts of the cell samples which had beenwithdrawn and by means of flow-cytometric determination carried out onthe human thrombocytes, which had been fixed and permeabilized asdescribed above. The autoradiograms of the Western blots were analyzedquantitatively using NIH gel-blotting image 1.6 software. The increasein antibody 16C2/phosphoserine 239 VASP binding (% increase (5 minvalue=maximum effect=100%) is recorded in Table 1.

TABLE 1 % Increase Thrombocytes Cell extract t (min) Cytometry Westernblotting 0 0 0 1 62.5 83.5 3 89.3 95.2 5 100.0 100.0

Example 6 Use of Antibody 16C2 to Analyze the Phosphorylation ofRecombinant VASP by cAMP-dependent Protein Kinase or cGMP-dependentProtein Kinase

Purified, recombinant hexahistidine-labeled VASP (25 μg/ml) wasphosphorylated by a purified catalytic subunit (11 μg/ml) of cAMPprotein kinase (CAPK) or by purified cGMP protein kinase (cGPK; 24μg/ml). Aliquots were removed from the reaction mixture at the giventimes, mixed immediately with SDS-containing stop solution, boiled andfractionated by means of SDS-PAGE. Phosphorylation of the VASP wasanalyzed by Coomassie Blue staining and by Western blotting using eithera polyclonal VASP antibody (M4, Halbrügge M. etal. J. Biol. Chem. 265,3088-3093 (1990), obtainable from:Alexis Corporation, AlteHauensteinstraβe 4, CH-4448 Läufelfingen, Switzerland) or the monoclonalVASP antibody 16C2. The band below VASP in the Coomassie Blue stainingis cAPK, while that above VASP is cGMP-PK The SDS-PAGE which wasperformed demonstrated the change in the migratory behavior of VASP from46 to 50 kDA due to the phosphorylation of serine 157, which ispreferred by 20 cAPK. The phosphorylation of serine 239 (detected by the16C2 antibody) is preferred by cGMP-PK.

Example 7 Analysis of the Phosphorylation, in Ptk2 Cells, of TransfectedVASP Containing Various Phosphorylation Mutations

The phosphorylation of transfected human VASP and VASP mutants wasinvestigated in Ptk2 cells. Wild-type VASP and VASP mutants each ofwhich contained mutated (inactivated) phosphorylation sites (alterationof serine 157, serine 238 or threonine 277), were transfected into Ptk2cells together with human cGMP protein kinase 1β and expressed under thecontrol of the CMV promoter. Ptk2 cells harbor very little endogenousVASP and cGMP protein kinase. Two days after transfection, the cellswere incubated for 30 min with 30 μM 8pCPT-cGMP and cell extracts werethen isolated. The phosphorylation of VASP in these cell extracts wasthen investigated in Western blots using the polyclonal antibody M4 andthe monoclonal antibody 16C2. A phosphorylation-dependent change of VASPfrom 46 kDa to 50 kDa was no longer present when serine 157 wasinactivated (mutation S157A). A signal recognized by antibody 16C2 wasno longer present when serine 239 was inactivated (S239A). In theseanalyses, mutations of threonine 277 behaved like wild-type VASP.

Example 8 Analysis of the Phosphorylation of VASP in Human ThrombocytesFollowing Stimulation with Various Vasodilators and cAMP/cGMP Analogs

Washed human thrombocytes (0.7×10⁹ cell/ml) were incubated with 1 μMPg-I₂ (prostacyclin), 0.5 mM 5,6-DCI-cBIMPS (cell membrane-permeablecAMP analog), 10 μM SNP (sodium nitroprusside) or 1 mM 8pCPTcGMP (cellmembrane-permeable cGMP analog). Aliquots (2.8×10⁷ cells) were removedafter the given times, mixed with the SDS stop solution and boiled, andthen investigated for VASP phosphorylation using the Western blottingmethod. The analyses were carried out using the polyclonal antibody M4or the monoclonal antibodies 16C2. Serine 157 phosphorylation wasquantified by the shift of VASP from 46 to 50 kDa, while serine 239phosphorylation was quantified by means of the signal given by the 16C2antibody.

Example 9 Phosphorylation of VASP in rat Thrombocytes

Rat thrombocytes (0.7×10⁹ cell/ml) were incubated for 5 min with 100 μMsodium nitroprusside (SNP), with 10 μM prostaglandin E1(PgE1) or withouteither of these additions. The extracts of these rat thrombocytes wereanalyzed by Western blotting. The blot demonstrated that the 5phosphorylation-dependent shift of VASP from 46 kDa to 50 kDa (serine157 phosphorylation) and the phosphorylation-dependent signal given bythe 16C2 antibody (serine 239 phosphorylation) also took place in ratthrombocytes. Similar results are also obtained using mousethrombocytes.

Example 10 Immunofluorescence Investigations of VASP and Phospho-VASPCarried out on Human Thrombocytes

Human thrombocytes (in platelet-rich plasma, PRP) were deposited on aglass slide and were allowed to attach and spread for 45 min. Thesethrombocytes were then incubated on the glass slide for 15 min without(A and C) or with 100 μM 8pCPTcGMP (B and D). The cells were thenimmediately fixed with 4% paraformaldehyde and permeabilized with 0.2%Triton X-100. They were then incubated with the polyclonal antibody M4(1:1000) or the monoclonal antibody 16C2, followed by the usualsecondary antibodies. Photographs show the appearance of thephosphorylation-dependent signal when the 16C2 antibody is used, whereasthe signal with the polyclonal M4 antibody (in the immunofluorescence,the sum of 46 kDa and 50 kDa VASP since there is no fractionation) isphosphorylation-independent.

What is claimed is:
 1. An antigen binding protein which only binds VASP(vasodilator-stimulated phosphoprotein) as antigen when VASP is presentin phosphorylated form, wherein said VASP is in the natural form or is afunctionally equivalent moiety, mutant, fragment or variant thereof andwherein the antigen binding protein is monoclonal antibody 16C2 which isproduced by the hybridoma cell line 16C2 deposited at the DeutscheSammiung von Mikroorganismen und Zellkulturen under the No. DSM ACC2330.2. The hybridoma cell line 16C2 (DSM ACC2330), which produces themonoclonal antibody of claim
 1. 3. A diagnostic kit, comprising anantigen binding protein which only recognizes VASP as antigen when VASPis present in phosphorylated form, wherein said VASP is in the naturalform or is a functionally equivalent moiety, mutant, fragment or variantthereof and wherein the antigen binding protein is monoclonal antibody16C2 which is produced by the hybridoma cell line 16C2 deposited at theDeutsche Sammiung von Mikroorganismen und Zeilkulturen under the No. DSMACC2330.